Defects in cardiac excitability are the basis for human arrhythmia and sudden cardiac death, a leading cause of mortality in developed countries. Ion channels and transporters control the movement of charged ions across cell membranes. In the heart, the coordinate activities of these proteins regulate the transmembrane electrochemical gradient to control depolarization/repolarization, and thus cardiac excitability. Normal function of ion channels and transporters requires defined biophysical properties as well as precise expression, organization, and regulation in defined membrane domains. Findings generated during our first period of funding support a new paradigm for human cardiac disease (arrhythmia) based on dysfunction in proteins that are required for proper expression and local regulation of ion channels and transporters at specific excitable membranes. Specifically, we discovered that ankyrin proteins, previously considered static membrane adapters, play dynamic roles in ion channel, transporter, and signaling protein targeting in ventricular cardiomyocytes. Patients harboring loss-of-function mutations in the ankyrin-B gene (ANK2) display a severe and complex cardiac phenotype. Phenotypes may include sinus node dysfunction, atrial fibrillation (AF), conduction defects, catecholamine-induced polymorphic ventricular arrhythmia, and/or sudden cardiac death. Moreover, we have learned that common ANK2 gene variants in the general population are associated with QTc alterations and ventricular arrhythmia susceptibility, that AnkB levels are strongly altered in large animal models of cardiovascular disease, and that the ANK2 is a candidate gene for AF susceptibility in the general human population. However, despite these translational studies implicating AnkB as a key player in cardiac excitability, the specific molecular roles of AnkB in heart remain surprisingly unknown. In fact, the identities of the in vivo cellular components of the AnkB-targeting pathway (or other cardiac targeting pathways) are still unknown. Finally, lack of an animal model of AnkB deficiency (global AnkB k/o is embryonic lethal) has prevented efforts to define new roles of AnkB in cardiac physiology and disease. For this first competitive renewal, due to important advances during the first funding cycle and the development of a number of innovative new animal models, we are well-positioned to provide the first in vivo information on the fundamental components (both upstream &downstream) of the entire AnkB-targeting pathway at baseline and in disease. We provide exciting new preliminary data that identifies a novel family of membrane trafficking proteins (EHD proteins) that regulate cardiac membrane excitability and associate with AnkB. We further provide new data that AnkB plays a novel role in targeting select Ca2+ channels in sinus node &atria. Finally, our preliminary data in mice demonstrates novel and unexpected roles of AnkB in cardiac membrane biogenesis and maintenance. Together, our published findings and preliminary data support a central hypothesis that the AnkB-based cellular pathway plays dynamic roles in myocyte membrane excitability and cardiac function. The immediate goals of our research program are to understand the specific cellular role(s) of AnkB in the heart (including upstream regulatory pathways [EHD proteins] and novel downstream targets [Cav1.3]) and determine how AnkB dysfunction leads to complex human cardiac disease. For this first competitive renewal, we present a cast of uncharacterized and innovative animal models, novel molecular tools, innovative technologies, and new antibodies to test the specific roles of the AnkB cellular pathway in vivo. PUBLIC HEALTH RELEVANCE: Normal function of ion channels/transporters requires defined biophysical properties as well as precise expression, organization, and regulation in defined membrane domains. Ankyrin-B is a critical regulator of membrane protein targeting in heart and ankyrin-B dysfunction results in severe human arrhythmia. Our new studies will provide insight on key upstream and downstream roles of AnkB in diverse excitable myocytes at baseline and in cardiovascular disease using a host of novel animal models.